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Involvement of Regulatory T Cells in the Experimental Autoimmune Encephalomyelitis-Preventive Effect of Dendritic Cells Expressing Myelin Oligodendrocyte Glycoprotein plus TRAIL¹

Department of Immunogenetics, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
We previously reported the protection from myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) by the adoptive transfer of genetically modified embryonic stem cell-derived dendritic cells (ES-DC) presenting MOG peptide in the context of MHC class II molecules and simultaneously expressing TRAIL (ES-DC-TRAIL/MOG). In the present study, we found the severity of EAE induced by another myelin autoantigen, myelin basic protein, was also decreased after treatment with ES-DC-TRAIL/MOG. This preventive effect diminished, if the function of CD4⁺CD25⁺ regulatory T cells (Treg) was abrogated by the injection of anti-CD25 mAb into mice before treatment with ES-DC-TRAIL/MOG. The adoptive transfer of CD4⁺CD25⁺ T cells from ES-DC-TRAIL/MOG-treated mice protected the recipient mice from MOG- or myelin basic protein-induced EAE. The number of Foxp3⁺ cells increased in the spinal cords of mice treated with ES-DC-TRAIL/MOG. In vitro experiments showed that TRAIL expressed in genetically modified ES-DC and also in LPS-stimulated splenic macrophages had a capacity to augment the proliferation of CD4⁺CD25⁺ T cells. These results suggest that the prevention of EAE by treatment with ES-DC-TRAIL/MOG is mediated, at least in part, by MOG-reactive CD4⁺CD25⁺ Treg propagated by ES-DC-TRAIL/MOG. For the treatment of organ-specific autoimmune diseases, induction of Treg reactive to the organ-specific autoantigens by the transfer of DC-presenting Ags and simultaneously overexpressing TRAIL therefore appears to be a promising strategy.

	Introduction

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For the treatment of subjects with autoimmune and allergic diseases, it is desirable to down-modulate the immune response in an Ag-specific manner while not causing systemic immune suppression. To achieve this goal, genetically modified dendritic cells (DC)⁴ presenting target Ags and simultaneously expressing immunoinhibitory molecules would be an attractive strategy (1).

TRAIL, a member of the TNF superfamily, is expressed in a variety of cell types, including lymphocytes, NK cells, NKT cells, and virus-infected APCs (2, 3, 4, 5). The abrogation of functional TRAIL by gene targeting or the in vivo administration of soluble death receptor 5, one of receptors for TRAIL, results in the acceleration of autoimmune diseases in mouse models, for example collagen-induced arthritis, autoimmune diabetes, and experimental autoimmune encephalomyelitis (EAE) (6, 7, 8, 9). It is thus evident that TRAIL plays a critical role in the regulation of the immune response or the maintenance of immunological self-tolerance to prevent autoimmunity. However, the precise mechanism for this has not yet been clarified regarding how TRAIL exerts such an effect.

We recently reported the protection from myelin oligodendrocyte glycoprotein (MOG)-induced EAE with genetically modified DC expressing MOG peptide along with TRAIL or programmed death-1 ligand (PD-L1) (1). For the genetic modification of DC, we used a method to generate DC from mouse embryonic stem cells in vitro (ES-DC) (10, 11, 12, 13). For the efficient presentation of MOG peptide in the context of MHC class II molecules, we used an expression vector in which cDNA encoding for human MHC class II-associated invariant chain was mutated to contain antigenic peptide in the class II-associated invariant chain peptide region (14, 15). An epitope inserted into this vector is efficiently presented in the context of coexpressed MHC class II molecules. Based on these technologies, we generated transfectant ES-DC presenting MOG peptide and simultaneously expressing TRAIL or PD-L1, ES-DC-TRAIL/MOG, and ES-DC-PDL1/MOG, respectively.

The treatment of mice with either of the double-transfectant ES-DC significantly reduced the severity of MOG-induced EAE. In contrast, treatment with ES-DC expressing MOG alone, irrelevant Ag (OVA) plus TRAIL, or OVA plus PD-L1, or coinjection with ES-DC expressing MOG plus ES-DC expressing TRAIL or PD-L1, had no effect on the disease course. The immune response to irrelevant exogenous Ag (keyhole limpet hemocyanin) was not impaired by treatment with any of the genetically modified ES-DC. These results suggest the possibility of treating autoimmune diseases without affecting immunity to exogenous Ags using genetically engineered DC presenting target autoantigen and simultaneously expressing TRAIL or PD-L1. In that study, we observed an increase in apoptosis of CD4⁺ T cells in the spleens of mice treated with ES-DC-TRAIL/MOG, suggesting that protection from EAE by ES-DC-TRAIL/MOG is mediated by induction of apoptosis of MOG-reactive pathogenic CD4⁺ T cells. In the present study, we found that the severity of not only MOG- but also myelin basic protein (MBP)-induced EAE was reduced by treatment with ES-DC-TRAIL/MOG. Regarding the mechanism underlying this disease-preventive effect, we obtained several lines of evidence supporting that MOG-reactive CD4⁺CD25⁺ regulatory T cells (Treg) were activated or propagated by the transfer of ES-DC-TRAIL/MOG and that the prevention of EAE by treatment with ES-DC-TRAIL/MOG was mediated, at least in part, by Treg.

	Materials and Methods

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Mice and cells

CBA and C57BL/6 mice obtained from Clea Animal or Charles River Laboratories were kept under specific pathogen-free conditions. Male CBA and female C57BL/6 mice were mated to generate F₁ (CBF₁) mice and all in vivo experiments were done using CBF₁ mice, syngeneic to TT2 ES cells. The mouse experiments were approved by the Animal Research Committee of Kumamoto University.

The mouse ES cell line, TT2, derived from CBF₁ blastocysts, and OP9 were maintained as previously described (10). The induction of differentiation of ES cells into ES-DC and generation of transfectant ES-DC-TRAIL, ES-DC-PDL1, ES-DC-MOG, ES-DC-TRAIL/MOG, ES-DC-PDL1/MOG, and ES-DC-TRAIL/OVA was done as described previously (1).

Protein, cytokines, and Abs

The mouse MOG p35–55 (MEVGWYRSPFSRVVHLYRNGK) and mouse MBP p35-47 (TGILDSIGRFFSG) were synthesized using the F-moc method on an automatic peptide synthesizer (PSSM8; Shimadzu) and purified using HPLC. Recombinant mouse GM-CSF (PeproTech) was purchased. Rat anti-mouse CD25 mAb was produced by culturing the PC61.5.3 cell line in the CELLINE system (BD Biosciences) and was purified by the MAbTrap kit (Amersham Biosciences). Abs and reagents used for staining were PE-conjugated anti-mouse CD25 (clone 3C7, rat IgG2b; BD Pharmingen) and FITC-conjugated anti-mouse CD4 (clone GK1.5, rat IgG2b; BD Pharmingen).

Induction of EAE and treatment with ES-DC

For the induction of EAE, 6- to 8-wk-old female CBF₁ mice were immunized by performing a s.c. injection at the base of tail with a 0.2-ml IFA/PBS solution containing 600 µg of MOG p35–55 peptide, MBP p35–47 peptide, or whole bovine MBP (Sigma-Aldrich), and 400 µg of Mycobacterium tuberculosis H37Ra (Difco Laboratories) on day 0. In addition, 500 ng of purified Bordetella pertussis toxin (Calbiochem) was injected i.p. on days 0 and 2 (1). For the prevention of EAE, mice were injected i.p. with ES-DC (1 x 10⁶ cells/mouse/injection) on days –8, –5, and –2 (preimmunization treatment), or on days 14, 17, and 21 (postonset treatment). In some experiments, CD25⁺ T cells were depleted by i.p. injections of anti-mouse CD25 mAb (clone PC61.5.3) as described (16). In brief, the mAb (400 µg/mouse) was administrated on days –28, –24, –21, and –14. Depletion was verified by staining PBMC and then analyzing them on a flow cytometer (FACScan; BD Biosciences). The mice were observed over a period of 42 or 56 days (postonset treatment) for clinical signs and scores were assigned based on the following scale: 0, normal; 1, weakness of the tail and/or paralysis of the distal half of the tail; 2, loss of tail tonicity and abnormal gait; 3, weakly partial hind-limb paralysis; 3.5, strongly partial hind-limb paralysis; 4, complete hind-limb paralysis; 5, fore-limb paralysis or moribundity; 6, death.

Adoptive transfer of T cells

For the adoptive transfer experiments, donor CBF₁ mice were i.p. injected with ES-DC (1 x 10⁶ cells/injection/mouse) on days –10, –7, and –4. CD4⁺ T cells and CD4⁺CD25⁺ T cells were isolated from the spleen cells of donor mice using the MACS cell sorting system (Miltenyi Biotec). For the isolation of CD4⁺ T cells, non-CD4⁺ T cells magnetically labeled with a biotin-conjugated Ab mixture (anti-CD8, anti-CD11b, anti-CD45R, anti-DX5, and anti-Ter-119) and anti-biotin microbeads were depleted on an autoMACS cell separator. Subsequently, CD25⁺ T cells, labeled with anti-CD25 mAb conjugated with PE and anti-PE microbeads, were isolated from the CD4⁺ T cell fraction using positive sorting columns. Cell purity was checked by FACS analysis: CD4⁺ T cells were >95% after the first step and CD4⁺CD25⁺ cells were >95% after the second step. The CD4⁺ T cells, CD4⁺CD25⁺ T cells, or CD4⁺CD25^– T cells were i.v. injected into recipient mice (2.5 x 10⁶, 3 x 10⁵, or 2.2 x 10⁶ cells/mouse, respectively) on day –2. The recipient mice were subjected to EAE induction (on days 0 and 2) as described above.

Proliferation assay of Treg

Mouse CD4⁺CD25⁺ Treg were purified with a Treg separation kit (MACS) from the spleen cells of naive CBF₁ mice as described above. Assay for the proliferation of Treg was done, as described previously (17). In brief, 1 x 10⁴ ES-DC or syngeneic splenic macrophages were x-ray irradiated (25 Gy), and cocultured with 1 x 10⁴ Treg in the presence of anti-CD3 mAb (clone 145-2C11, 1 µg/ml) and human IL-2 (10–30 U/ml) in wells of 96-well round-bottom culture plates. In some assay, anti-TRAIL mAb (clone N2B2 (5 µg/ml); eBioscience) was added. Splenic macrophages were prepared by collecting plastic dish-adherent cells. The cells were cultured for 3 days, and [³H]thymidine (6.7 Ci/mM) was added to the culture (1 µCi/well) in the last 12 h. At the end of culture, cells were harvested onto glass fiber filters (Wallac) and the incorporation of [³H]thymidine was measured by scintillation counting. The expression of TRAIL in LPS-stimulated spleen cells and macrophages was confirmed by RT-PCR, as described previously (1). The relative quantity of cDNA in each sample was first normalized by PCR for G3PDH. The primer sequences were as follows: TRAIL, 5'-AACCCTCTAGACCGCCGCCACCATGCCTTCCTCAGGGGCCCTGAA-3' and 5'-GAAATGGTGTCCTGAAAGGTTC; G3PDH, 5'-GGAAAGCTGTGGCGTGATG-3' and 5'-CTGTTGCTGTAGCCGTATTC-3'.

Immunohistochemical analysis

Freshly excised spinal cords were immediately frozen and embedded in Tissue-Tek OCT compound (Sakura Fine Technical). Immunohistochemical staining of Foxp3 and CD4 was done, as previously described (1, 12), but with some modification. In brief, serial 7-µm sections were made using cryostat and underwent immunochemical staining with anti-Foxp3 mAb (clone FJK-16s, rat IgG2a; eBioscience) or CD4 (clone L3T4; BD Pharmingen), and N-Histofine Simple Stain Mouse MAX PO (Nichirei).

Statistical analysis

The two-tailed Student’s t test was used to determine any statistical significance of differences. A value of p < 0.05 was considered to indicate statistical significance (15). The values indicated in tables are rounded off to the first decimal place.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Prevention of MBP-induced EAE by the transfer of ES-DC genetically engineered to express MOG peptide along with TRAIL

We recently demonstrated the prevention of MOG-induced EAE by treatment with ES-DC expressing MOG peptide plus TRAIL (ES-DC-TRAIL/MOG) or MOG peptide plus PD-L1 (ES-DC-PDL1/MOG). Regarding the mechanism for preventing EAE by the genetically modified ES-DC, we considered not only the possibility of the direct down-modulation of MOG-reactive effector T cells such as the induction of anergy or apoptosis, but also the possibility of promoting MOG-reactive T cells with regulatory or immune-suppressive functions. We hypothesized that, if the latter had been the case, then pretreatment with ES-DC-TRAIL/MOG or ES-DC-PDL1/MOG may thus have had some preventive effect on not only MOG- but also MBP-induced EAE. To test this possibility, we pretreated mice with ES-DC-TRAIL/MOG or ES-DC-PDL1/MOG and subjected them to EAE induction by immunization with MBP (whole protein) or MBP p35-47, according to the schedule depicted in Fig. 1A. As a result, we found the severity of both MBP whole protein- and peptide-induced EAE to be significantly reduced by pretreatment with ES-DC-TRAIL/MOG. In contrast, pretreatment with ES-DC-PDL1/MOG, ES-DC-TRAIL/OVA (as irrelevant Ag), and ES-DC-MOG had no effect on MBP-induced EAE (Fig. 1, B–D, and Table I).

View larger version (21K): [in this window] [in a new window]   FIGURE 1. Prevention of MBP-induced EAE by treating the mice with ES-DC-TRAIL/MOG. A, The schedule for the pretreatment and induction of EAE is shown. CBF1 mice (three to four mice per group) were i.p. injected with ES-DC (1 x 106 cells/mouse/injection) on days –8, –5, and –2. EAE was induced by the immunization of MBP peptide or whole protein on day 0, and the injection of B. pertussis toxin on days 0 and 2. B–D, The disease severity of mice immunized with MBP peptide (B) or whole protein (C and D) is shown. The data of all experiments are summarized in Table I.

View larger version (17K): [in this window] [in a new window]   FIGURE 2. Inhibition of MBP-induced EAE by treating the mice with ES-DC-TRAIL/MOG. A, The schedule for induction of EAE and treatment is shown. CBF1 mice (five to seven mice per group) were immunized on days 0 and 2 according to the EAE-induction schedule described above, and subsequently i.p. injected with ES-DC (1 x 106 cells/mouse/injection) on days 14, 17, and 21. B, The disease severity of mice immunized with MBP whole protein is shown.

We considered the possibility that MOG-reactive T cells possessing some immunoregulatory effect were activated or propagated by the transferred ES-DC-TRAIL/MOG and that the T cells exerted a protective effect against MBP-induced EAE. To address this possibility, we performed adoptive transfer experiments. We isolated CD4⁺ T cells from the spleens of the donor mice treated with ES-DC and transferred them into naive recipient mice. Subsequently, the recipient mice were subjected to an immunization procedure for MOG-induced EAE (Fig. 3A). As shown in Fig. 3, B and C, and Table II, the transfer of 2.5 x 10⁶ CD4⁺ T cells isolated from the mice treated with ES-DC-TRAIL/MOG significantly reduced the severity of EAE of the recipient mice. In contrast, CD4⁺ T cells isolated from the mice treated with ES-DC-PDL1/MOG or ES-DC-MOG or those from untreated mice showed no effect. These results support the notion that CD4⁺ T cells with some regulatory activity were induced, activated, or propagated by the treatment with ES-DC-TRAIL/MOG.

View larger version (20K): [in this window] [in a new window]   FIGURE 3. Protection from MOG or MBP-induced EAE by the adoptive transfer of CD4+CD25+ T cells from the mice treated with ES-DC expressing MOG plus TRAIL. A, The schedule for the treatment of donor mice with ES-DC, the adoptive transfer of some population of T cells, and the induction of EAE in the recipient mice are shown. The donor CBF1 mice were i.p. injected with ES-DC (1 x 106 cells/mouse/injection) on days –10, –7, and –4. The T cells were isolated from the donor mice and then transferred to naive mice on day –2. The recipient mice were immunized with MOG peptide (B–D) or MBP whole protein (E and F) on days 0 and 2 according to the EAE-induction protocol as shown in Fig. 1. B and C, The disease severity of the mice transferred with CD4+ T cells (2.5 x 106 cells/mouse) from ES-DC-TRAIL/MOG, ES-DC-PD-L1/MOG, ES-DC-MOG, ES-DC-TRAIL/OVA-treated mice, or naive mice is shown. D–F, The disease severity of the mice transferred with CD4+CD25+ T cells or CD4+CD25– T cells (3 x 105 or 2.2 x 106 cells/mouse, respectively) from ES-DC-TRAIL/MOG-treated mice or naive mice is shown. Control mice were injected with RPMI 1640 medium alone without T cells. The data of all experiments are summarized in Table II.

We next tested whether the transfer of CD4⁺CD25⁺ T cells isolated from donor mice treated with ES-DC-TRAIL/MOG would have any effect on MBP-induced EAE. We transferred CD4⁺CD25⁺ T cells from the donor mice treated with ES-DC as described above. Subsequently, the recipient mice were subjected to an immunization procedure for MBP-induced EAE. As shown in Fig. 3, E and F, and Table II, the transfer of 3 x 10⁵ CD4⁺CD25⁺ T cells isolated from mice treated with ES-DC-TRAIL/MOG significantly reduced the severity of MBP-induced EAE in the recipient mice, but the transfer of CD4⁺CD25⁺ T cells isolated from ES-DC-TRAIL/OVA-treated mice or naive mice did not do so. These results indicate that ES-DC-TRAIL/MOG-induced CD4⁺CD25⁺ T cells had a protective effect against not only MOG- but also MBP-induced EAE.

Protection from MBP-induced EAE by ES-DC-TRAIL/MOG depends on CD25⁺ cells

To further verify the involvement of CD4⁺CD25⁺ T cells in the disease-preventive effect of ES-DC-TRAIL/MOG, we performed depletion experiments of CD4⁺CD25⁺ T cells. We injected the mice with anti-mouse CD25 mAb four times on days –28, –24, –21, and –14 (Fig. 4A). Depletion of CD4⁺CD25⁺ T cells was confirmed by flow cytometry analysis of PBLs. CD25⁺ cells were 0.3 ± 0.1% of CD4⁺ T cells in anti-CD25 mAb-treated mice (n = 4), whereas they were 2.6 ± 0.3% in control (PBS-injected) mice (n = 3). Representative results of flow cytometry analysis are shown in Fig. 4B. Thereafter, we treated the mice with ES-DC-TRAIL/MOG and then immunized them with MBP according to the EAE induction protocol. As shown in Fig. 4C and Table I, the effect of ES-DC-TRAIL/MOG to prevent MBP-induced EAE completely disappeared in the mice in which the CD4⁺CD25⁺ T cells were depleted. These results further support the possibility that the prevention of MBP-induced EAE by ES-DC-TRAIL/MOG was mediated by CD4⁺CD25⁺ T cells. In addition, treatment with anti-CD25 mAb slightly worsened the disease course even when mice were not treated with ES-DC, suggesting that CD4⁺CD25⁺ T cells ameliorate the disease to some extent in the natural course after EAE induction.

View larger version (22K): [in this window] [in a new window]   FIGURE 4. The depletion of CD4+CD25+ T cells diminished the preventive effect of ES-DC-TRAIL/MOG on MBP-induced EAE. A, The schedule for the depletion with anti-CD25 mAb, pretreatments with ES-DC, and induction of EAE are shown. CBF1 mice (three to four mice per group) were i.p. injected with anti-mouse CD25 mAb (400 µg/mouse/injection) on days –28, –24, –21, –14 and then were treated with ES-DC (1 x 106 cells/mouse/injection) on days –8, –5, and –2. EAE was induced by immunization with MBP whole protein on days 0 and 2 as in Fig. 1. B, CD4+CD25+ cells of peripheral blood of anti-CD25 mAb-treated (right) and PBS-treated control (left) mice were analyzed by flow cytometry on day –2. The percentage of CD4+CD25+ cells among CD4+ cells is shown. C, The severity of MBP-induced EAE is shown. Control mice were injected with PBS alone without Ab and RPMI 1640 medium alone without ES-DC. The data of all experiments are summarized in Table I.

At present, Foxp3 is the most reliable molecular marker for Treg (18). We performed immunohistochemical analysis to detect Foxp3⁺ cells in the spinal cord of mice treated with ES-DC. We treated the mice with ES-DC and then immunized them with MOG according to the EAE induction protocol. On day 11, the spinal cords harvested from mice were stained with anti-Foxp3 mAb (Fig. 5, A–C) and anti-CD4 mAb, and Foxp3⁺ cells and CD4⁺ cells were counted. As shown in Fig. 5D, the infiltration of Foxp3⁺ cells into the spinal cord was enhanced in mice treated with ES-DC-TRAIL/MOG, compared with mice with no treatment or mice treated with ES-DC-PDL1/MOG. In contrast, the infiltration of CD4⁺ cells into the spinal cords was reduced in mice treated with ES-DC-TRAIL/MOG or ES-DC-PDL1/MOG, compared with the observation in mice with no treatment. These results further support the notion of involvement of Treg in the disease-protection effect.

View larger version (30K): [in this window] [in a new window]   FIGURE 5. Increased number of Foxp3+ cells in spinal cords of mice treated with ES-DC expressing MOG plus TRAIL. CBF1 mice (five mice per group) were pretreated with ES-DC-TRAIL/MOG or PDL1/MOG (1 x 106 cells/mouse/injection) as in Fig. 1 or left untreated. Subsequently, EAE was induced by immunization with MOG peptide on days 0 and 2. The cervical, thoracic, and lumbar spinal cord was isolated on day 11 and subjected to immunohistochemical analysis. The Foxp3+ cells (A–C, arrowhead) and CD4+ cells were stained and microscopically counted in three sections of spinal cord (D). Results are expressed as mean of samples obtained from five mice ± SD. *, The increase in number of infiltrated cells is statistically significant (p < 0.01) as compared with other groups.

Recent studies have demonstrated that bone marrow-derived DC and splenic DC have a potent capacity to promote the proliferation of CD4⁺CD25⁺ Treg both in vitro and in vivo (17, 19). We investigated whether ES-DC had the capacity to promote the proliferation of CD4⁺CD25⁺ Treg and also whether the expression of TRAIL had any effect on this capacity of ES-DC. Treg isolated from spleen of naive CBF₁ mice were cocultured with ES-DC in the presence of anti-CD3 mAb (1 µg/ml) and a low dose of human IL-2 (10 U/ml). As shown in Fig. 6A, all three types of ES-DC, nontransfectant ES-DC, ES-DC-TRAIL, or ES-DC-PDL1, induced a proliferation of Treg more potently than splenic macrophages. The increased proliferation of Treg was observed upon coculture with ES-DC-TRAIL, in comparison to that with other type of ES-DC. In contrast, the magnitude of proliferation of CD4⁺CD25^– conventional Th cells was decreased by the expression of TRAIL by ES-DC (Fig. 6B). In addition, anti-TRAIL blocking mAb decreased the proliferation of Treg cocultured with ES-DC-TRAIL (Fig. 6C). These results suggest that TRAIL expressed on ES-DC has an inhibitory effect on the proliferation of conventional CD4⁺ T cells and a stimulating effect on that of CD4⁺CD25⁺ Treg.

View larger version (24K): [in this window] [in a new window]   FIGURE 6. Enhanced capacity of APC expressing TRAIL to induce the proliferation of naive CD4+CD25+ Tregs in vitro. A, CD4+CD25+ T cells (1 x 104) or (B) CD4+CD25– T cells (1 x 104) isolated from the spleen of the naive CBF1 mice were cultured alone or cocultured with irradiated stimulators (1 x 104), syngeneic splenic macrophages, nontransfectant ES-DC (TT2), ES-DC-TRAIL, or ES-DC-PDL1, for 3 days in the presence of anti-CD3 mAb (0.1 µg/ml) and human IL-2 (10 U/ml). C, CD4+CD25+ T cells (1 x 104) isolated from the spleen of the naive CBF1 mice were cultured alone or cocultured with irradiated stimulators (1 x 104), nontransfectant ES-DC (TT2), or ES-DC-TRAIL, for 3 days in the presence of anti-CD3 mAb (2.5 µg/ml) and human IL-2 (10 U/ml) with or without anti-mouse TRAIL mAb (2.5 µg/ml). D, The expression of TRAIL in spleen cells or macrophages stimulated with LPS was confirmed by RT-PCR. The naive Treg isolated from a spleen were cocultured for 3 days with 1 x 105 of irradiated LPS-stimulated spleen cells (E) or 1 x 104 of irradiated LPS-stimulated macrophages (F) in the presence of anti-CD3 mAb (1 µg/ml) and IL-2 (30–100 U/ml) with or without anti-mouse TRAIL mAb (5 µg/ml) for 3 days. The proliferation of responder T cells was quantified by measuring the [3H]thymidine incorporation in the last 12 h of the culture. *, Statistical significance (p < 0.01). The results were expressed as the mean of a triplicate assay ± SD. The data are each representative of more than three independent experiments with similar results.

The data presented so far suggest that TRAIL expressed on ES-DC has an effect to augment proliferation of CD4⁺CD25⁺ Treg. Lastly, we investigate the effect of TRAIL expressed on natural APCs on proliferation of Treg. Recent studies reported that LPS-stimulation enhanced the expression of TRAIL on spleen cells and bone marrow-derived DC (20, 21, 22). We thus examined the proliferation of Treg cocultured with LPS-stimulated whole spleen cells or splenic macrophages in the presence or absence of anti-TRAIL-blocking mAb. Treg isolated from spleen of naive CBF₁ mice were cocultured with LPS-treated APC in the presence of anti-CD3 mAb and a low dose of human IL-2 with or without anti-TRAIL mAb (5 µg/ml). As shown in Fig. 6, D–F, anti-TRAIL mAb partially decreased the proliferation of CD4⁺CD25⁺ Treg. These results indicate that TRAIL naturally expressed on APC as well as that expressed on genetically modified ES-DC has a stimulating effect on proliferation of CD4⁺CD25⁺ Treg.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
In the present study, we found that the severity of not only MOG- but also MBP-induced EAE was reduced by treatment with ES-DC expressing MOG peptide along with TRAIL (Figs. 1 and 2). We obtained several lines of evidences suggesting a possibility that the observed disease-preventive effect is mediated by propagation of MOG-reactive Treg by ES-DC-TRAIL/MOG. Another possible explanation for this phenomenon may be as follows: MOG-reactive T cells might be activated by so-called epitope spreading even in MBP-induced EAE and they might play a major role in the pathogenesis; pretreatment with ES-DC-TRAIL/MOG may abrogate MOG-reactive T cells, thus resulting in a reduction of the disease severity. However, we consider this possibility to be less likely because ES-DC-PDL1/MOG, which showed a MOG-induced EAE-preventive effect similar to that induced by ES-DC-TRAIL/MOG, had no effect on MBP-induced EAE (Fig. 1, B and C, and Table I). In addition, in previous studies of EAE, the autoreactivity directed to multiple myelin Ags caused by epitope spreading have been observed in chronic or relapsing phases over 4 wk after the immunization. In contrast, the inhibitory effect on MBP-induced EAE, which we observed in the present study, occurred within 2 wk after the immunization. We therefore considered the possibility that MOG-specific T cells with some regulatory activity may have been induced or stimulated by ES-DC-TRAIL/MOG. The results of adoptive transfer experiments demonstrating that CD4⁺ T cells isolated from ES-DC-TRAIL/MOG-treated donor mice acted to protect the recipient mice from EAE (Fig. 3, A–C, and Table II) strongly support this notion.

So far, several types of T cells involved in the negative regulation of immune responses have been identified, such as IL-10-producing Tr1 cells and CD4⁺CD25⁺ Treg (17, 19, 23). Kohm et al. (24) reported that the adoptive transfer of relatively large number of CD4⁺CD25⁺ Treg (2 x 10⁶) isolated from naive mice protected the recipient mice from MOG-induced EAE. It was recently reported that immature DC induced Tr1 cells producing high amounts of IL-10 (25), and also that the proliferation of CD4⁺CD25⁺ Treg was efficiently promoted by DC (17). We thus attempted to characterize the T cells with regulatory activity induced by ES-DC expressing TRAIL and involved in the protection from EAE. We quantified IL-10, IFN-, and IL-4 produced by spleen cells isolated from ES-DC-treated mice upon in vitro stimulation with MOG peptide, by ELISA. No significant change in the amount of these cytokines produced by spleen cells of ES-DC-TRAIL/MOG, ES-DC-PDL1/MOG, or ES-DC-MOG-treated mice was observed (data not shown). We thus considered it less likely that the disease-prevention effect was mediated by IL-10-producing Tr1 cells or Th2 cells, although we cannot totally rule out this possibility.

We next assessed the possibility that ES-DC-TRAIL/MOG had propagated or activated CD4⁺CD25⁺ Treg in vivo. Adoptive transfer experiments showed the presence of CD4⁺CD25⁺ T cells with a capacity to prevent not only MOG- but also MBP-induced EAE in ES-DC-TRAIL/MOG-treated mice (Fig. 3, D–F, and Table II). In addition, when CD4⁺CD25⁺ T cells were depleted by the pretreatment of mice with anti-CD25 mAb, the protective effect of ES-DC-TRAIL/MOG against MBP-induced EAE was totally abrogated (Fig. 4 and Table I), the observation further supporting the notion that CD4⁺CD25⁺ T cells play a role in the prevention of MBP-induced EAE by treatment with ES-DC-TRAIL/MOG. Recently, Kohm et al. (26) reported that the effect of anti-CD25 Ab is not the depletion but functional inactivation of Treg. Our findings that preventive effect of MBP-induced EAE with ES-DC-TRAIL/MOG was diminished by the injection of anti-CD25 mAb into mice also may be interpreted as inactivation of Treg. Several groups recently reported that CD4⁺CD25^– T cells converted to Treg functionality in particular condition (27, 28). It is possible that CD4⁺CD25⁺ Tregs suppressing EAE observed in our study were also those converted from conventional CD4⁺ T cells.

Furthermore, we observed an increased number of Foxp3⁺ cells and the ratio of Foxp3⁺ cells to CD4⁺ cells in the spinal cords in mice treated with ES-DC-TRAIL/MOG (Fig. 5). In contrast, we could not detect any significant increase in the expression of Foxp3 mRNA in the spleen of ES-DC-TRAIL/MOG-treated mice (data not shown). Also in flow cytometry analysis the proportion of Foxp3⁺ cells to CD4⁺ cells in the spleen and inguinal lymph nodes was not increased in mice treated with ES-DC-TRAIL/MOG as compared with control mice (the proportion of Foxp3⁺ cells to CD4⁺ cells was 9.7 ± 0.7% in spleen of control mice, 9.8 ± 0.8% in spleen of ES-DC-TRAIL/MOG-treated mice, 9.2 ± 0.9% in inguinal lymph nodes of control mice, and 9.5 ± 0.1% in inguinal lymph nodes of ES-DC-TRAIL/MOG-treated mice). Probably the increase in the number of MOG-reactive Foxp3⁺ cells was too small to be detected in total CD4⁺ T cells of spleen and inguinal lymph nodes. In addition, to investigate whether the ES-DC-TRAIL/MOG were acting at the level of priming or within the target organ (CNS), we tested the primary proliferative response to MBP of the T cells of mice treated with ES-DC-TRAIL/MOG before immunization with MBP for EAE induction (as shown in Fig. 1A). The inguinal lymph node cells were harvested on day 19 after the immunization and cultured in the presence of MBP (whole protein, 0, 12.5, 25, 50 µg/ml) for 3 days. In this experiment, the treatment with ES-DC-TRAIL/MOG did not reduce the proliferative response to MBP of T cells in inguinal lymph node (data not shown). This result suggests that treatment with ES-DC-TRAIL/MOG did not act at the level of priming. It may be more likely that the treatment with ES-DC-TRAIL/MOG acted in the target organ (CNS), as shown in Fig. 7.

View larger version (45K): [in this window] [in a new window]   FIGURE 7. Promotion of Tregs with DC expressing TRAIL plus certain tissue-specific autoantigen as a novel strategy for the treatment of the organ-specific autoimmune diseases. DC expressing TRAIL and simultaneously presenting known tissue-specific autoantigen (auto-Ag) in the context of their MHC class II can activate or propagate T cells with a regulatory function (Tr cells) reactive to the autoantigen. In the focus of autoimmune disease, the promoted-Tr cells were retriggered by the same autoantigen expressed in the tissue. Then, the Tr cells inhibited the pathogenic T cells responding to unknown autoantigen. This strategy is applicable, even if target autoantigens are unidentified or if multiple Ags are recognized as targets by the pathogenic autoreactive T cells.

Consistent with recent reports on the stimulation of CD4⁺CD25⁺ Treg as well as conventional T cells by splenic or BM-derived DC (17, 29), in vitro experiments showed that ES-DC also have the capacity to induce proliferation of both CD4⁺CD25^– conventional T cells and CD4⁺CD25⁺ Treg, when stimulated with anti-CD3 mAb in the presence of low-dose IL-2. The expression of TRAIL on ES-DC enhanced the capacity to induce proliferation of CD4⁺CD25⁺ Treg, but not of CD4⁺CD25^– conventional T cells (Fig. 6, A and B). In addition, anti-TRAIL-blocking mAb decreased the proliferation of Treg cocultured with ES-DC-TRAIL or natural APC, such as LPS-treated spleen cells or macrophage (Fig. 6, C–F). The results of these in vitro experiments suggest that the in vivo transfer of ES-DC-TRAIL/MOG induced proliferation of MOG-reactive Treg which protected the recipient mice from EAE.

Based on the results obtained in the current study, we consider that the inhibition of autoimmunity by TRAIL-expressing ES-DC may be attributed to the promotion of CD4⁺CD25⁺ T cells by TRAIL, in addition to the induction of apoptosis of pathogenic T cells as suggested by our previous study (1). Mi et al. (30) reported that TRAIL inhibited the proliferation of diabetogenic T cells isolated from NOD mice by suppressing IL-2 production and up-regulating the expression of p27^kip1. It may be possible that the effects of Treg were involved in their observations. In addition, Herbeuval et al. (31) reported that a level of TRAIL was elevated in plasma of HIV-1-infected patients and in vitro exposure to HIV-1 induced the expression of TRAIL in APCs. Andersson et al. (32) reported expression of Foxp3 to be enhanced in the lymphoid tissue of HIV-infected patients. These two findings may also be related to our findings.

DC modified by some way to enhance their tolerogenic characteristics is regarded as a promising therapeutic means to negatively manipulate the immune response for the treatment of autoimmune and allergic diseases and also for the induction of transplantation tolerance (1, 23, 33, 34, 35, 36). In the clinically manifest phase of autoimmune diseases, such as multiple sclerosis or type I diabetes, it is presumed that multiple tissue-specific Ags are recognized as targets by deregulated immunity due to epitope spreading (37). As a result, the induction of a mere deletion or anergy of pathogenic T cells specific to primarily recognized autoantigens may not be sufficient to control these diseases. The promotion of the immune-suppressive T cells reactive to organ-specific self Ags by treatment with genetically modified DC may be a promising therapeutic modality for subjects with autoimmune diseases (Fig. 7). This strategy may also be useful for the induction of transplantation tolerance.

	Acknowledgments

 
We thank Dr. S. Aizawa (RIKEN Center for Developmental Biology, Kobe, Japan) for TT2, Drs. N. Takakura (Kanazawa University, Kanazawa, Japan) and T. Suda (Keio University, Tokyo, Japan) for OP9, Dr. H. Niwa (RIKEN Center for Developmental Biology, Kobe, Japan) for pCAG-IPuro, Drs. T. Okazaki and T. Honjo (Kyoto University, Kyoto, Japan) for a cDNA clone for PD-L1, and Drs. T. Koda and T. Nishimura (Hokkaido University, Sapporo, Japan) for a cDNA clone for OVA.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported in part by Grants-in-Aid 12213111, 14370115, 14570421, 14657082, and the Program of Founding Research Centers for Emerging and Reemerging Infectious Disease from the Ministry of Education, Science, Technology, Sports, and Culture, Japan, and a Research Grant for Intractable Diseases from the Ministry of Health, Labour and Welfare, Japan, and grants from the Uehara Memorial Foundation, and by funding from the Meiji Institute of Health Science.

² Y.N. and S.S. contributed equally to this study.

³ Address correspondence and reprint requests to Dr. Satoru Senju, Department of Immunogenetics, Graduate School of Medical Sciences, Kumamoto University, Honjo 1-1-1, Kumamoto 860-8556, Japan. E-mail address: senjusat{at}gpo.kumamoto-u.ac.jp

⁴ Abbreviations used in this paper: DC, dendritic cell; EAE, experimental autoimmune encephalomyelitis; MOG, myelin oligodendrocyte glycoprotein; ES, embryonic stem cell; PD-L1, programmed death-1 ligand; MBP, myelin basic protein; Treg, regulatory T cell; Trl, T regulatory type 1.

Received for publication March 9, 2006. Accepted for publication October 31, 2006.

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